Use of a Fibrinogen Capture Agent to Detect a Ciz1 B-Variant

ABSTRACT

The present invention relates to the use of a fibrinogen capture agent or a fibrinogen detection agent in an assay to detect a Ciz1 b-variant.

FIELD OF THE INVENTION

The present invention relates to assays and methods useful for thedetection of Cip1-interacting zinc finger protein (Ciz1) in a sample. Inparticular, the present invention relates to the use of such assays andmethods for the diagnosis of cancer.

BACKGROUND OF THE INVENTION

Cip1-interacting zinc finger protein 1 (Ciz1) (as exemplified by NCBIReference Sequence: NM_001131016.1) is required for cell proliferation.Ciz1 localises to nuclear matrix bound foci that form sites of DNAreplication during early S phase and promotes the initiation of DNAreplication in association with cell cycle regulators including cyclinA/cyclin dependent kinase (CDK)2, cyclin E/CDK2 and p21cip1. In thecontext of transcription, CIZ1 is an oestrogen responsive gene that isitself a positive cofactor of the oestrogen receptor (ER), capable ofenhancing the recruitment of ER to target chromatin. Ciz1 isalternatively spliced to produce conserved isoforms in mouse and man.Normal Ciz1 protein comprises at least two defined functional domains, a‘replication’ domain and an ‘immobilisation’ domain.

Alternative splicing of Ciz1 exon 14 to generate a Ciz1 b-variant hasbeen determined in various cancers, including small cell lung cancer(SCLC), non-small cell lung cancer (NSCLC), lymphomas, thyroid, kidneyand liver cancer. Excess expression of either the replication orimmobilisation domain of Ciz1 has also been demonstrated in cancers, forexample in NSCLC, breast, colon, kidney, liver, bladder and thyroidcancers. A correlation of domain expression with the stage of the cancerhas also been determined (see WO 2012/078208).

The present invention addresses the continued need to develop diagnostictests and treatments that improve the survival rates of patientssuffering from cancers such as lung cancer through novel biomarkers andtargets.

SUMMARY OF THE INVENTION

The present inventors have determined that Ciz1 b-variant exists in acomplex with fibrinogen. This has allowed the inventors to develop anovel assay for detecting the presence of Ciz1 b-variant in a subject.

Furthermore, the present inventors have determined that Ciz1 b-variantmay exist as peptide fragments. Furthermore, the present inventors havedetermined that Ciz1 b-variant and/or fibrinogen/Ciz1 b-variantcomplexes may exist in the exosomal compartment of a blood sample.

These findings instruct the design of assays for the detection of Ciz1b-variant peptides, either when alone or when in complex withfibrinogen.

In particular, the finding that Ciz1 b-variant is complexed withfibrinogen has enabled the provision of a detection assay, in particulara diagnostic assay, which utilises the capture of fibrinogen or Ciz1b-variant as an initial step in order to isolate the fibrinogen/Ciz1b-variant complex. The assay may involve the use a fibrinogen captureagent to isolate the fibrinogen/Ciz1 b-variant complex followed by theuse of Ciz1 b-variant detection agent to detect the Ciz1 b-variant whichmay be within said isolated complex. Alternatively, the assay mayinvolve the use a Ciz1 b-variant capture agent to isolate thefibrinogen/Ciz1 b-variant complex followed by the use of fibrinogendetection agent to detect the fibrinogen which may be within saidisolated complex.

Thus, in a first aspect the present invention relates to the use of afibrinogen capture agent or a fibrinogen detection agent in an assay todetect a Ciz1 b-variant.

As used herein, a capture agent refers to an agent which is used toisolate (i.e. enrich) the fibrinogen/Ciz1 b-variant complex from asample. For example, the sample may be a sample which has been isolatedfrom a subject. In particular, the sample may be a blood sample (e.g. aplasma sample) which has been isolated from a subject.

As used herein, a detection agent refers to an agent which is used tobind the fibrinogen/Ciz1 b-variant complex after it has been isolated(i.e. enriched) from a sample using a capture agent. As such, thedetection agent is typically used after a capture step to determine thepresence of the isolated (i.e. enriched) fibrinogen/Ciz1 b-variantcomplex.

Suitably, the assay may be an enzyme-linked immunosorbent assay.

The fibrinogen capture agent or fibrinogen detection agent may be anantibody or a fragment thereof, non-protein scaffold, aptamer or Ciz1b-variant peptide which specifically binds fibrinogen.

In a preferred embodiment, the fibrinogen capture agent or fibrinogendetection agent is an antibody or a fragment thereof which specificallybinds fibrinogen. In another preferred embodiment, the fibrinogencapture agent is a Ciz1 b-variant peptide which specifically bindsfibrinogen.

The fibrinogen may be fibrinogen alpha chain.

The Ciz1 b-variant may be detected in a sample from a subject, forexample a blood, urine, saliva or bronchoalveolar lavage sample.

In a preferred embodiment the sample is a blood sample from a subject.In a particularly preferred embodiment the sample is a plasma sample.

The Ciz1 b-variant may be detected by a method which comprises thefollowing steps: a) capturing a fibrinogen/Ciz1 b-variant complex usinga fibrinogen capture agent; and b) detecting Ciz1 b-variant using a Ciz1b-variant detection agent; or a) capturing a fibrinogen/Ciz1 b-variantcomplex using a Ciz1 b-variant capture agent; and b) detectingfibrinogen using a fibrinogen detection agent.

The Ciz1 b-variant capture agent may be an antibody or a fragmentthereof, or a non-protein scaffold which specifically binds Ciz1b-variant.

The Ciz1 b-variant capture agent may be an antibody or a fragmentthereof which specifically binds Ciz1 b-variant.

In a further aspect the present invention relates to a method ofdiagnosing cancer in a subject which comprises detecting a Ciz1b-variant peptide in a sample from the subject, wherein the presence ofCiz1 b-variant peptide in the sample indicates that the subject hascancer and wherein the method comprises the step of capturing afibrinogen/Ciz1 b-variant peptide complex from the sample.

In a further aspect the present invention relates to a method ofdiagnosing cancer in a subject which comprises detecting a Ciz1b-variant peptide in a sample from the subject, wherein the presence ofCiz1 b-variant peptide in the sample indicates that the subject hascancer and wherein the Ciz1 b-variant peptide is the sequence shown asSEQ ID NO: 11 or a fragment thereof which comprises EVR as shown inpositions 36 to 38 of SEQ ID NO: 11.

In another aspect the present invention relates to method of detecting aCiz1 b-variant peptide in a sample, for example detecting a Ciz1b-variant peptide in a sample from a subject, wherein the methodcomprises the step of capturing a fibrinogen/Ciz1 b-variant peptidecomplex from the sample.

The method may comprise the steps of capturing a fibrinogen/Ciz1b-variant complex using a fibrinogen capture agent; and detecting Ciz1b-variant using a Ciz1 b-variant detection agent; or capturing afibrinogen/Ciz1 b-variant complex using a Ciz1 b-variant capture agent;and detecting fibrinogen using a fibrinogen detection agent.

Suitably, the step of detecting the Ciz1 b-variant peptide or fibrinogenmay employ antibody-based arrays, enzyme linked immunosorbent assays(ELISA), non-antibody protein scaffolds, radioimmuno-assay (RIA),western blotting, aptamers or mass spectrometry.

The method may further comprise the step of releasing a Ciz1 b-variantpeptide and/or a fibrinogen/Ciz1 b-variant peptide complex from anexosomal component of the sample.

The Ciz1 b-variant peptide and/or fibrinogen/Ciz1 b-variant peptidecomplex may be released from the exosomal component by detergenttreatment, such as SDS treatment.

The method may further comprise the step of enriching the exosomalfraction from the sample.

For example, the step of enriching the exosomal fraction may compriseultracentrifugation, ultrafiltration, continuous flow electrophoresis,chromatography or cross-flow ultrafiltration.

The cancer may be selected from lung, lymphoma, kidney, breast, liver,bladder, ovarian or thyroid cancer.

In particular, the cancer may be lung cancer.

The present invention further relates to a method for treating a subjectwith cancer comprising administering to the subject a cancertherapeutic; wherein the subject has been identified as having cancer bythe method of the invention.

The present invention also provides an anticancer drug for use intreating cancer wherein the subject has been identified as having cancerby the method of the invention.

The present invention further provides an anticancer lung cancer drugfor use in treating lung cancer wherein the subject has been identifiedas having lung cancer by the method of the invention.

In a further aspect the present invention provides the use of adetergent to release a Ciz1 b-variant and/or a fibrinogen/Ciz1 b-variantcomplex from an exosomal compartment of a sample.

In another aspect the present invention relates to a method fordetecting a Ciz1 b-variant in a sample which comprises the step ofreleasing the Ciz1 b-variant and/or a fibrinogen/Ciz1 b-variant complexfrom an exosomal compartment of the sample by treating the sample withdetergent and detecting if the Ciz1 b-variant is present in the sample.The method may further comprise the step of enriching the exosomalfraction from the sample prior to releasing the Ciz1 b-variant.

The step of enriching the exosomal fraction may compriseultracentrifugation, ultrafiltration, continuous flow electrophoresis,chromatography or cross-flow ultrafiltration.

In a further aspect the present invention provides a kit comprising anagent which specifically binds fibrinogen and an agent whichspecifically binds Ciz1 b-variant.

The fibrinogen may fibrinogen alpha chain.

The agent which specifically binds fibrinogen and/or the agent whichspecifically binds Ciz1 b-variant may be an antibody or a fragmentthereof, a non-protein scaffold or an aptamer.

The agent which specifically binds to fibrinogen may be a Ciz1 b-variantpeptide.

According to any of the above aspects of the present invention, the Ciz1b-variant may be a Ciz1 b-variant peptide shown as SEQ ID NO: 11 or afragment thereof which comprises EVR as shown in positions 36 to 38 ofSEQ ID NO: 11.

DESCRIPTION OF THE DRAWINGS

FIG. 1—a) Ciz1 b-variant epitope-containing species, detected withsplice junction-selective anti-Ciz1 rabbit polyclonal antibody 043. 043was generated and validated as described for antibody 2B (Higgins etal.; (2012); PNAS; Nov 6; 109(45):E3128-35) and is capable of detectionof Ciz1 b-variant by western blot in plasma from lung cancer patients.Western blots were performed and show Ciz1 b-variant in 0.5 ul ofrepresentative samples from 5 patients with lung carcinomas (variousstages) and five from individuals with no disease. b) Separation ofplasma from lung cancer patients into the exosomal compartment andsoluble compartment illustrated here using Invitrogen Total ExosomeIsolation Kit (Catalog Number 4484450) used as recommended by themanufacturer. Fractions were separated by SDS-PAGE, and probed with 043to reveal Ciz1 b-variant, or with ‘generic’ Ciz1 antibodies thatrecognizes an epitope elsewhere in the protein (data shown here isgenerated with NB100-74624 (Nov4), Novus Biologicals that detects theC-terminus of Ciz1). Lane 1, untreated plasma (0.5 ul), lane 2 after lowspeed clearing spin (0.5 ul), lane 3 after high speed clearing spin (0.5ul), lane 4 after addition of exosome reagent (0.5 ul equivalent), lane5 high speed spin supernatant (SN, 0.5 ul equivalent), lane 6 high speedspin pellet (0.5 ul equivalent), lane 7 high speed spin pellet (P, 1 ulequivalent), lane 8 high speed spin pellet (2 ul equivalent). Red arrowsindicate key lanes, showing different partitioning of Ciz1 epitopes. M,molecular weight marker. c) Comparison of type 1 plasma samples in whichCiz1 b-variant is exclusively in the exosome fraction, and rare patientswho display a type 2 pattern in which a fraction of Ciz1 b-variant is inthe soluble fraction. d) Quantitative immuno-analysis generated using aprototype sandwich ELISA (without treating samples with detergent)showing increased signal in the type 2 plasma sample illustrated in c.e) Partitioning of Ciz1 b-variant in naïve type 1 plasma, and aftertreatment with 3% SDS (tested in the presence of protease inhibitor, PI,or 10 mM EDTA), revealing a shift of epitope into the soluble fraction.Blue arrows indicate soluble fraction after application of exosomeisolation protocol. MW markers are shown to the right. SN=supernatant,P=pellet (exosome). f) Enhanced cancer-specific signal in SDS-treatedtype 1 plasma in ELISA immunoassay.

FIG. 2—Ciz1 Sequences

FIG. 3—Ciz1 b-variant ˜65-70 kDa species is a Ciz1 b-variant peptidestably bound to a carrier protein, which may augment antigenicity ofCiz1 b-variant peptide. a) Western blot illustrating cancer-selectiveepitope recognised by anti-Ciz1 b-variant antibody 2B, as describedpreviously (Higgins et al; as above). b) Reconstitution of a Ciz1b-variant epitope from synthetic b-variant peptide (50 nmols ofEIAGQDEDHFITVDAVGCFEGDEEEEEDDEDEEEIEVRSRDISREEWKGSETYSPNTAYGV DFLV—SEQID NO: 12) mixed with 0.5 ul of non-cancer plasma. Free peptide migrateswith mobility indicative of a MW 3-4× greater than predicted (˜30 kDainstead of 7.6 kDa), indicated by blue arrow. When incubated in plasmamobility decreases, antigenicity increases, and a new species is createdat ˜80 kDa, indicated by red arrow. c) Similar results were generatedwith a separate anti-Ciz1 b-variant monoclonal antibody. d) Forreference, a duplicate blot is probed with a monoclonal antibody that isselective for the exon 14a version of the peptide(EIAGQDEDHFITVDAVGCFEGDEEEEEDDEDEEEIEVEEELCKQRSRDISREEWKGSETYSPNTAYGVDFLV—SEQ ID NO: 13), showing detection of the peptide in lanes 1and 3. Key: Lane 1, WT peptide only. Lane 2, B-var peptide only. Lane 3,WT peptide+normal plasma. Lane 4, B-var peptide+normal plasma. Lane 5,normal plasma only. Results show that normal plasma acquires Ciz1b-variant signal at ˜80 kDa upon addition of b-var peptide, but nota-var peptide. e) Similar results were obtained using short b-variantpeptide (DEEEIEVRSRDIS—SEQ ID NO: 2), when combined with either canceror non-cancer plasma. Western blot shows results with anti-Ciz1b-variant monoclonal antibody (left) or anti-Ciz1 b-variant polyclonalantibody 043 (right). Key: Lane 1, B-var peptide only. Lane 2, B-varpeptide+normal plasma. Lane 3, B-var peptide+lung cancer plasma. Lane 4,Normal plasma alone. Lane 5, Lung cancer plasma alone. Note thatmobility of new signal is similar to endogenous signal in cancer patientplasma. F) Western blot showing titration of short peptide(DEEEIEVRSRDIS—SEQ ID NO: 2) into cancer patient plasma, detected withanti-Ciz1 b-variant monoclonal antibody (upper) or anti-Ciz1 b-variantpolyclonal antibody 043 (lower). Graph shows quantification of resultsby densitometry, with signal in normal plasma (N) set at 1. Results showthat carrier protein is not limiting up to 4 nmol peptide/0.5 ul plasma.Overall, results show that endogenous b-var65 species, detected by antiCiz1 b-variant antibodies, is likely not one single 65 kDa Ciz1b-variant species, but a stable composite of Ciz1 b-variant fragment andcarrier protein with MW close to 65 kDa.

FIG. 4—Isolation using a two-dimensional gel approach shows that thecarrier protein is Fibrinogen. a) Western blot of cancer (C) andnon-cancer (N) plasma samples after incubation in 2% SDS sample bufferwith and without 200 mM b-mercaptoethanol. In the presence of reducingagent, a cancer-specific signal at ˜65 kDa is produced with anti-bvariant antibodies, while in the absence of reducing agentcancer-specific signal is retarded to ˜340 kDa. b) First dimension gel(without reducing agent) is used to isolate Ciz1 b-variantepitope-containing ˜340 kDa band. c) Band is further resolved afterincubation in reducing agent to reveal constitutive polypeptides andmigration of Ciz1 b-variant epitope with a ˜70 kDa component. The bandwas excised for mass spectrometry. d) MASCOT assigned one significantidentity based on two separate peptide masses after trypsin digest.Identity: Fibrinogen alpha chain.

FIG. 5—Reconstitution of Ciz1 b-variant epitope from purified fibrinogenand synthetic peptides. a) Silver stained gel (left) showing 6 nmols ofpurified fibrinogen complex (Sigma Cat. F3879), after electrophoresis inthe absence of reducing agent (in 2% SDS loading buffer). Peptides areincluded in the experiment (100 pmols/lane) but do not stain well withsilver due to their amino-acid content. Western blots of parallel gelsare shown on the right, probed with two anti-b variant antibodies. L,Long Ciz1 b-variant peptideEIAGQDEDHFITVDAVGCFEGDEEEEEDDEDEEEIEVRSRDISREEWKGSETYSPNTAYGV DFLV (SEQID NO: 12). S, Short Ciz1 b-variant peptide DEEEIEVRSRDIS (SEQ ID NO:2). b) Similar experiment comparing a range of Ciz1 b-variant anda-variant peptides for their ability to form epitope that is reactive tothe same antibodies, when in complex with fibrinogen. Key: 1, noadditions. 2, Long Ciz1 b-variant peptideEIAGQDEDHFITVDAVGCFEGDEEEEEDDEDEEEIEVRSRDISREEWKGSETYSPNTAYGV DFLV (SEQID NO: 12). 3, WT long peptideEIAGQDEDHFITVDAVGCFEGDEEEEEDDEDEEEIEVEEELCKQRSRDISREEWKGSETYSPNTAYGVDFLV (SEQ ID NO: 13). 4, no additions. 5, Short Ciz1 b-variantDEEEIEVRSRDIS (SEQ ID NO: 2). 6, C-DEEγIEVRSRDIS-coNH2 (SEQ ID NO: 14).7, C-DEγEIEVRSRDIS-coNH2 (SEQ ID NO: 23). 8, C-DγγγIγVRSRDIS-coNH2 (SEQID NO: 15).

FIG. 6—Table 1: Ciz1 b-variant peptides comprising an exon 14b/exon 15junction (specified by exclusion of VEEELCKQ—SEQ ID NO: 10) from NCBIReference Sequence: NM_001131016.1.

FIG. 7—Exon junction specific antibodies detect CIZ1b in lung cancerpatient plasma. A) Map of CIZ1 translated exons showing alternativesplicing of exon 14 to yield 14b (CIZ1b) by exclusion of the indicated 8amino-acids (positions refer to NCBI Reference Sequence: NM_012127.2).Also shown are the location of epitopes detected with antibodies used inB. B) Western blot of representative plasma samples from a patient withNSCLC (LC) and normal control (N) after denaturation for SDS-PAGE,showing CIZ1b band at 65-70 kDa in LC (red arrow, marked “*”) detectedby CIZ1b antibodies 2B and 043, and a ‘generic’ CIZ1 antibody-reactiveband at 55 kDa in both N and LC, detected via epitopes in exon 8 andexon 17. Also indicated is fibrinogen alpha chain. C) Comparison ofCIZ1b levels in paired plasma (P) and serum (S) samples from fourindividuals analysed with CIZ1b antibody 2B. Histogram shows quantifiedband intensities for the 65-70 kDa and 55 kDa entities, derived fromtriplicate samples, with standard deviation. Note that serum lacks the65-70 kDa CIZ1b band. D) Model showing the simplest interpretation ofthe data in B, though extensive alternative splicing and likelysecondary modification mean that the exact identity of the 55 kDa entityin plasma is unverified. E) Receiver operating characteristic (ROC)curves showing area under the curve (AUC) values for 2B and 043 westernblot results on lung cancer test set A. Dot plot shows correlationbetween the two date sets (LC samples are orange). Thresholds (blackdotted lines) are set at the mean of non-cancer samples in the set, plusone SD. These yield sensitivity/specificity estimates for this set of 20lung cancer and 20 non-cancer samples of: 2B, 85/67.5; 043, 90/77.5.Using an arbitrary threshold specified by both antibodies these are90/87.5.

FIG. 8—Effect of denaturation on CIZ1b epitope. A) Left, Coomassie bluestained gel of a representative lung cancer (C) and normal (N) plasmasample (2 ul), separated under fully native conditions (in the absenceof SDS or reducing agent). m indicates marker lanes. Middle, parallelgel after transfer to nitrocellulose stained with Ponceau S. Right, thesame membrane probed with CIZ1b antibody 043. Lower panel shows the sametwo samples (red arrows) separated by denaturing SDS-PAGE, also probedwith 043. B) Native gel first dimension was soaked in 4×SDS-PAGE loadingbuffer for 30 minutes without heating, and further separated by size(second dimension). Western blot reveals one band at 55 kDa that isreactive with antibody 2B in the non-cancer sample, and two bands (55and 70 kDa) in the cancer sample. Two additional cancer plasma samples,treated in the same way are shown below. C) Migration of 043-reactivebands in a representative cancer (C) and non-cancer (N) plasma samplethrough a non-denaturing gel, after prior incubation at the indicatedtemperatures, in the presence of 2% SDS, with and without reducing agent(200 mM β-mercaptoethanol, βME) as indicated. Note the shift ofcancer-specific band (red box) from relative mobility of approximately340 kDa in the absence of reducing agent, to ˜70 kDa in the presence ofreducing agent (accompanied by complete loss of the ‘generic’ band thatwas detected in all samples in the absence of reducing agent). D) Plasmafrom a lung cancer patient showing 043-reactive band after incubationwith 1% SDS at 37° C. for 30 mins, with the indicated concentrations ofDTT, or a 10-fold excess of βME. The reactive band is indicated as itshifts from 340 kDa to 70 kDa, and is then lost under maximally reducingconditions. The behaviour of fibrinogen in the same samples is shown forcomparison, detected with antibody F8512. E) Synthetic CIZ1b epitopegenerated by combining CIZ1b peptide (b66, Table 2) with normal(non-cancer) plasma (see FIG. 10), behaves similarly to endogenousepitope, dissociating from within a high molecular weight complex, to a˜70 kDa species, and then disappearing under maximally reducingconditions. Fibrinogen is detected with antibody AF4786. βMEconcentrations refer to multiples of the standard concentration used inSDS-PAGE of 200 mM.

FIG. 9—Deconstruction of CIZ1b biomarker and immunoassay design A)Schematic of results shown in FIG. 8, illustrating the complexity of thenative complex in which CIZ1b epitope (red bar, marked “*”) resides inplasma, and the disrupting effect of detergent and reducing agents.Under native conditions CIZ1b is part of a complex in excess of 720 kDa,most likely encompassed within lipid vesicles. SDS shifts the epitope to340 kDa, and reducing agents shift it further to the mobility typicallyobserved in standard SDS-PAGE gels (65-70 kDa). In the presence ofexcess reducing agent the western blot epitope is lost for both 2B and043 CIZ1b antibodies. B) Schematic of sandwich ELISA format based oninformation summarised in A.

FIG. 10—Reconstitution of CIZ1b biomarker from purified components A)Two independent lung cancer patient plasmas were separated afterincubation with 1% SDS (no reducing agent), in order to isolate the 340kDa band from stained gels (left). Band identity was verified by ponceauS stain followed by Western blot with anti-CIZ1b antibody 043 of aparallel gel (right). B) Gel slices were soaked in 2% SDS-PAGE loadingbuffer (with 200 mM βME) and separated through a denaturing gel torecover the 043-reactive species at 70 kDa. Note that sample loaded as agel slice is slightly retardation compared to markers which are loadedin solution. Coomassie blue staining revealed 5 dominant bands includingone at 70 kDa, which was isolated and digested with either trypsin(cancer plasma 1, C1) or AspN (cancer plasma 2, C2). C) Western blot ofnormal human plasma (non-cancer, N) showing reconstitution of CIZ1bepitope via complex formation between a carrier protein in plasma andsynthetic CIZ1b peptides of the indicated lengths, but not equivalentCIZ1a peptide (Table 2). Note that free peptides migrate in reducingSDS-PAGE with a relative mobility 3 times expected (˜21 kDa instead of7.6 kDa for b66, ˜5 kDa instead of 1.6 for b13). None are recognized byCIZ1b antibodies in western blot unless complexed with carrier protein.For CIZ1b66 peptide a new reactive species is created at ˜80 kDa, whileCIZ1b13 peptide increases antigenicity at the same mobility as theendogenous epitope in lung cancer plasmas (C, lane 1). D) Western blotof non-reducing SDS-PAGE gel showing purified fibrinogen (6 nmols) inall lanes (upper), with and without prior incubation with 100 pmols ofthe indicated peptides (Table 2), probed with CIZ1b antibodies asindicated. Under non-reducing conditions fibrinogen migrates withapparent molecular weight of ˜340 kDa and this co-migrates with CIZ1bsignal. E) Direct ELISA showing mean A450 nm (triplicate analysis withSEM) generated by CIZ1b antibodies 2B or 043 as indicated, using molarequivalents of preformed peptide/fibrinogen complex as analyte. ** Ttest p<0.05.

FIG. 11—Immunoassay validation using lung cancer plasma and controls(n=39 non-cancer, 13 lung cancer). A) CIZ1b western blot with antibody043. B) Sandwich ELISA format for CIZ1b antigen in plasma, using 043capture antibody and anti-fibrinogen detector antibody. C) Quantitativedetection of fibrinogen in the same samples using paired fibrinogenantibodies D) Substitution of anti-human IgG for anti-fibrinogen in asandwich format similar to that shown in B. E) Detection of IgG inplasma by direct ELISA. F) Normalization of the data in C against thedata in G. In all cases, receiver operating characteristic curves (ROC)indicate the relationship between true positive and false positivefraction (black points) and 95% confidence interval of the fitted ROCcurve (grey points), and area under the curve (AUC) values in bold. Boxand whisker plots display minimum and maximum, lower, median, and upperquartile, and outliers.

DETAILED DESCRIPTION OF THE INVENTION Ciz1

Cip1-interacting zinc finger protein (Ciz1) is a protein that in humansis encoded by the CIZ1 gene.

Ciz1 promotes initiation of mammalian DNA replication, where it helpscoordinate the sequential functions of cyclin E- and A-dependent proteinkinases (Coverley et al; J Cell Sci. 2005; 118(Pt 1):101-112). Itinteracts directly with cyclins E and A, CDK2, and cyclin-dependentkinase inhibitor p21 and also plays an indirect role in DNA replicationby modulating the expression of genes, including cyclin D, thatinfluence cell proliferation (den Hollander et al; Cancer Res. 2006;66(22):11021-11029). Ciz1 is often attached to the salt- andnuclease-resistant protein component of the nucleus referred to as the“nuclear matrix” and resides within foci that partially colocalize withsites of DNA replication (Ainscough; J Cell Sci. 2007; 120(Pt1):115-124), implicating Ciz1 in the spatial organization of DNAreplication.

Various splice variants of Ciz1 are known in the art. For example, asdescribed in WO 2004/051269 and Rahman et al. (BMC Cancer; 2010;10:482).

The alternative splicing of Ciz1 exon 14 to generate a Ciz1 b-variantisoform has previously been demonstrated in various cancers ((WO2012/017208) and (Higgins et al.; PNAS; Nov 6; 109(45):E3128-35)).

As described herein, a Ciz1 b-variant isoform is derived from a Ciz1mRNA transcript which comprises a variant of exon 14 referred to hereinas exon 14b (SEQ ID NO: 3). Ciz1 exon 14b lacks 24 nucleotides at the 3′end as compared to full length exon 14, referred to as exon 14a (SEQ IDNO: 4). Ciz1 transcripts expressing exon 14b rather than exon 14a(a-variant) are referred to herein as Ciz1 b-variant, CIZ1b or simplyb-variant. The corresponding amino acid sequences of Ciz1 exon 14b and14a are shown as SEQ ID NO: 5(LKSLEKEIAGQDEDHFITVDAVGCFEGDEEEEEDDEDEEEIE) and SEQ ID NO: 6(LKSLEKEIAGQDEDHFITVDAVGCFEGDEEEEEDDEDEEEIEVEEELCKQ) respectively (seeFIG. 2).

The sequence spanning a splice junction of exon 14b and exon 15 istherefore different to the sequence spanning a splice junction of exon14a and exon 15. In particular, the junction of exon 14b and exon 15lacks the sequence VEEELCKQ (SEQ ID NO: 10) which is present at thejunction between exon 14a and exon 15 in Ciz1 a-variant proteins.

The present inventors have determined that Ciz1 b-variant peptides existin a complex with fibrinogen. In particular, it has been shown that Ciz1b-variant peptides which span an exon 14b/exon 15 junction are able tobind to fibrinogen. In contrast, Ciz1 a-variant peptides are not able tobind to fibrinogen.

Accordingly, as described herein, a Ciz1 b-variant peptide refers to apeptide which can be derived from a Ciz1 b-variant protein but not aCiz1 a-variant protein. In other words, a Ciz1 b-variant peptide refersto a portion of a Ciz1 b-variant protein which is derived from exons 14band exon 15 of a Ciz1 b-variant protein and therefore lacks the sequenceVEEELCKQ (SEQ ID NO: 10) at the splice junction.

In particular, a Ciz1 b-variant peptide comprises an exon 14b/exon 15splice junction. In other words, a ciz1 b-variant peptide comprisesamino acid sequences derived from Ciz1 exon 14b (SEQ ID NO: 5) and Ciz1exon 15 (SEQ ID NO: 7) and comprises the sequence EVR as shown inpositions 36 to 38 of SEQ ID NO: 11.

SEQ ID NO: 11 EIAGQDEDHFITVDAVGCFEGDEEEEEDDEDEEEIEVRSRDISREEWKGSETYSPNTAYGVDFLVP

Notably, Ciz1 b-variant peptides do not contain amino acids which arespecific to Ciz1 a-variant proteins and therefore do not comprise thesequence VEEELCKQ (SEQ ID NO: 10).

As such, a Ciz1 b-variant peptide may be the Ciz1 b-variant peptideshown as SEQ ID NO: 11 or a fragment thereof which comprises EVR asshown in positions 36 to 38 of SEQ ID NO: 11.

Suitably, the Ciz1 b-variant peptide may comprise at least 3, at least5, at least 10, at least 20, at least 30, at least 40, at least 50 or atleast 60 contiguous amino acids of SEQ ID NO: 11 and comprise EVR asshown in positions 36 to 38 of SEQ ID NO: 11.

Suitably, the Ciz1 b-variant peptide may comprise or consist of from 3to 60, 5 to 60, 5 to 50, 10 to 50, 10 to 40, 10 to 30, 15 to 30 or 10 to20 contiguous amino acids of SEQ ID NO: 11 and comprise EVR as shown inpositions 36 to 38 of SEQ ID NO: 11.

Suitably, the Ciz1 b-variant peptide may comprise or consist of 10 to 20contiguous amino acids of SEQ ID NO: 11 and comprise EVR as shown inpositions 36 to 38 of SEQ ID NO: 11.

Suitably, a Ciz1 b-variant peptide may be a peptide shown in Table 1(see FIG. 6).

Fibrinogen

In one aspect, the present invention relates to the use of a fibrinogencapture agent or a fibrinogen detection agent in an assay to detect Ciz1b-variant.

In one aspect, the present invention relates to the use of a fibrinogenbinding agent in an assay to detect Ciz1 b-variant.

The fibrinogen binding agent may be an antibody or a fragment thereof,non-protein scaffold or aptamer which specifically binds fibrinogen.

In particular, the present invention provides the use of an antibody ora fragment thereof, which binds fibrinogen in an assay to detect Ciz1b-variant.

Fibrinogen is involved in blood clotting, fibrinolysis, cellular andmatrix interactions, the inflammatory response, wound healing andneoplasia.

Fibrinogen molecules are elongated 45 nm structures that consist of twoouter D domains, each connected by a coiled-coil segment to a central Edomain. The molecule is comprised of two sets of three polypeptidechains termed α, β, and γ, which are joined together in the N-terminal Edomain by five symmetrical disulfide bridges. Non-symmetrical disulfidebridges form a ‘disulfide ring’ in this region (Mosesson; 2005; Journalof Thrombosis and Haemostasis; 3(8); 1894-1940).

Polymerisation of fibrinogen molecules results in the formation of aninsoluble fibrin matrix. Conversion of fibrinogen to fibrin is triggeredby thrombin, which cleaves fibrinopeptides A and B from α and β chains,and thus exposes the N-terminal polymerization sites responsible for theformation of the soft clot. The soft clot is converted into the hardclot by factor XIIIA which catalyzes the epsilon-(gamma-glutamyl)lysinecross-linking between γ chains (stronger) and between α chains (weaker)of different monomers.

The α-chain consists of 610, the β-chain 461, and the major γ-chainform, γA, 411 residues. Each fibrinogen α-chain contains an N-terminalfibrinopeptide A (FPA) sequence, cleavage of which by thrombin initiatesfibrin assembly by exposing a polymerization site termed EA. One portionof EA is at the N-terminus of the fibrin α-chain comprising residues17-20 gly-pro-arg-val (GPRV), and another portion is located in thefibrin β-chain between residues 15 and 42. Each EA-site combines with aconstitutive complementary-binding pocket (Da) in the D domain ofneighbouring molecules that is located between γ337 and γ379. Theinitial EA:Da associations cause fibrin molecules to align in astaggered overlapping end-to-middle domain arrangement to formdouble-stranded twisting fibrils. Fibrils also undergo lateralassociations to create multi-stranded fibres (Mosesson; as above).

An example fibrinogen α protein is the human fibrinogen α protein havingthe UniProtKB accession number P02671. This exemplified sequence is 866amino acids in length of which amino acids 1 to 19 form a signalpeptide.

An example fibrinogen β protein is the human fibrinogen β protein havingthe UniProtKB accession number P02675. This exemplified sequence is 491amino acids in length of which amino acids 1 to 30 form a signalpeptide.

An example fibrinogen γ protein is the human fibrinogen γ protein havingthe UniProtKB accession number P02679. This exemplified sequence is 453amino acids in length of which amino acids 1 to 26 form a signalpeptide.

The term ‘fibrinogen’ may refer to a complex comprising two α chains,two β chains and two γ chains or a sub-complex thereof (e.g. which doesnot include all the chains of the full fibrinogen complex).

The present inventors have determined that Ciz1 b-variant peptides arecapable of binding to the fibrinogen α chain. As such, in particularembodiments the present assays and methods may involve the capture ordetection of the fibrinogen α chain. For example the present assays andmethods may involve the detection of fibrinogen (e.g. fibrinogen αchain) following the capture of Ciz1 b-variant in the fibrinogen/Ciz1b-variant complex.

A ‘fibrinogen/Ciz1 b-variant complex’ as referred to herein may compriseother peptides and/or proteins aside from the fibrinogen and/or Ciz1b-variant peptide.

As used herein, a capture agent refers to an agent which is used toisolate (i.e. enrich) the fibrinogen/Ciz1 b-variant complex from asample. For example, the sample may be a sample which has been isolatedfrom a subject. In particular, the sample may be a blood sample (e.g. aplasma sample) which has been isolated from a subject.

A ‘fibrinogen capture agent’ refers to an entity which is capable ofbinding to a fibrinogen/Ciz1 b-variant complex. Preferably, a‘fibrinogen capture agent’ is an entity which is capable of specificallybinding to fibrinogen and is used to isolate (i.e. enrich) thefibrinogen/Ciz1 b-variant complex from a sample. Such specific bindingmay enable the isolation/enrichment of fibrinogen from a sample. Inparticular embodiments, the fibrinogen capture agent may be capable ofspecifically binding to a fibrinogen α chain.

The fibrinogen capture agent may be an antibody or a fragment thereof,non-protein scaffold, aptamer or Ciz1 b-variant peptide whichspecifically binds fibrinogen.

As used herein, a detection agent refers to an agent which is used tobind the fibrinogen/Ciz1 b-variant complex after it has been isolated(i.e. enriched) from a sample using a capture agent. As such, thedetection agent is typically used after a capture step to determine thepresence of the isolated (i.e. enriched) fibrinogen/Ciz1 b-variantcomplex.

A ‘fibrinogen detection agent’ refers to an entity which is capable ofbinding to a fibrinogen. Preferably, a ‘fibrinogen detection agent’ isan entity which is capable of specifically binding to fibrinogen. Suchspecific binding enables the detection of fibrinogen following thecapture of Ciz1 b-variant in the fibrinogen/Ciz1 b-variant complex. Inparticular embodiments, the fibrinogen capture agent or fibrinogendetection agent may be capable of specifically binding to a fibrinogen αchain.

The fibrinogen detection agent may be an antibody or a fragment thereof,non-protein scaffold, or aptamer which specifically binds fibrinogen.

The fibrinogen detection agent may be labelled with an entity whichenables it to be detected in an assay as described herein. For example,the fibrinogen detection agent may be labelled with a fluorescent entityor an enzyme-substrate label as described herein.

Antibody

An antibody or a fragment thereof, refers to any portion of an antibodywhich retains the ability to bind to the same antigen target as theparental antibody—e.g. a fibrinogen antibody or a fragment thereof isable to bind to fibrinogen.

Fibrinogen antibodies are known in the art. For example, sheepanti-fibrinogen pAb (AF4786 R&D systems), ab58207 (Abcam);Anti-Fibrinogen, clone 85D4 (Cat. No. F9902; Sigma Aldrich); andFibrinogen Antibody (MFB-HB) (Cat. No. MA1-35371; Life Technologies).

Fibrinogen α chain antibodies are also known in the art. For example,EPR2918 (ab108616; Abcam); Fibrinogen Antibody (5C5) (LF-MA0108;Pierce); and EPR2919 (TA307697; Origene).

The present invention also encompasses methods which comprise the stepof detecting Ciz1 b-variant or capturing Ciz1 b-variant/fibrinogencomplex by contacting the Ciz1 b-variant with an antibody or fragmentthereof that binds to the Ciz1 b-variant. An antibody which specificallybinds a Ciz1 b-variant peptide may be generated as previously describedfor antibody 2B (Higgins et al; 2012; PNAS; Nov 6; 109(45):E3128-35).

The antibody may be a chimeric antibody. Chimeric antibodies may beproduced by transplanting antibody variable domains from one species(for example, a mouse) onto antibody constant domains from anotherspecies (for example a human).

The antibody may be a full-length, classical antibody. For example theantibody may be an IgG, IgM or IgA molecule.

The antibody may be a functional antibody fragment. Specific antibodyfragments include, but are not limited to, (i) the Fab fragmentconsisting of VL, VH, CL and CH1 domains, (ii) the Fd fragmentconsisting of the VH and CH1 domains, (iii) the Fv fragment consistingof the VL and VH domains of a single antibody, (iv) the dAb fragment,which consists of a single variable domain, (v) isolated CDR regions,(vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fabfragments (vii) single chain Fv molecules (scFv), wherein a VH domainand a VL domain are linked by a peptide linker which allows the twodomains to associate to form an antigen binding site, (viii) bispecificsingle chain Fv dimers, and (ix) “diabodies” or “triabodies”,multivalent or multispecific fragments constructed by gene fusion. Theantibody fragments may be modified. For example, the molecules may bestabilized by the incorporation of disulphide bridges linking the VH andVL domains.

The antibody described herein may be a multispecific antibody, andnotably a bispecific antibody, also sometimes referred to as“diabodies”. These are antibodies that bind to two (or more) differentantigens. Diabodies can be manufactured in a variety of ways known inthe art, e.g., prepared chemically or from hybrid hybridomas. Theantibody may be a minibody. Minibodies are minimized antibody-likeproteins comprising a scFv joined to a CH3 domain. In some cases, thescFv can be joined to the Fc region, and may include some or all of thehinge region.

The antibody may be a domain antibody (also referred to as asingle-domain antibody or nanobody). This is an antibody fragmentcontaining a single monomeric single variable antibody domain. Examplesof single-domain antibodies include, but are not limited to, VHHfragments originally found in camelids and VNAR fragments originallyfound in cartilaginous fishes. Single-domain antibodies may also begenerated by splitting the dimeric variable domains from common IgGmolecules into monomers.

The antibody may be a synthetic antibody (also referred to as anantibody mimetic). Antibody mimetics include, but are not limited to,Affibodies, DARPins, Anticalins, Avimers, Versabodies and Duocalins.

Aptamers

Aptamers that specifically recognize fibrinogen or Ciz1 b-variant may besynthesized using standard nucleic acid synthesis techniques or selectedfrom a large random sequence pool, for example using the SystematicEvolution of Ligands by Exponential Enrichment (SELEX) technique.

Aptamers can be single strand DNA or RNA sequences that fold in a unique3D structure having a combination of stems, loops, quadruplexes,pseudoknots, bulges, or hairpins. The molecular recognition of aptamersresults from intermolecular interactions such as the stacking ofaromatic rings, electrostatic and van der Waals interactions, orhydrogen bonding with a target compound. In addition, the specificinteraction between an aptamer and its target is complemented through aninduced fit mechanism, which requires the aptamer to adopt a uniquefolded structure to its target. Aptamers can be modified to be linkedwith labeling molecules such as dyes, or immobilized on the surface ofbeads or substrates for different applications.

Aptamers may be paired with nanotechnology, microarray, microfluidics,mass spectrometry and other technologies for quantification in a givensample.

Typically, a fibrinogen capture agent or a Ciz1 b-variant capture agentwill be immobilised on a support, such as an array, or be captured on asolid support, such as beads. A sample to be tested is then incubatedwith the support comprising the capture agent such that Ciz1b-variant/fibrinogen complex can be isolated/enriched from the sample.Supports (eg. solid supports) can be made of a variety of materials—suchas glass, silica, plastic, nylon or nitrocellulose. When attached to asolid support it is preferably rigid and have a planar surface.

The fibrinogen capture agent or Ciz1 b-variant capture agent maycomprise a moiety that can be captured on a solid surface, such as abiotin moiety which can be captured by streptavidin beads. In suchembodiments, capture to a support can occur follow incubation of thecapture agent with a sample.

Detecting Ciz1 b-Variant or Capturing a Ciz1 b-Variant

Suitably, the Ciz1 b-variant may be a Ciz1 b-variant peptide asdescribed herein.

A Ciz1 b-variant may be detected using a variety of suitable methods andtechniques known in the art.

The method may comprise the following steps: a) capturing afibrinogen/Ciz1 b-variant complex using a fibrinogen capture agent; andb) detecting Ciz1 b-variant using a Ciz1 b-variant detection agent.

The method may comprise the following steps: a) capturing afibrinogen/Ciz1 b-variant complex using a Ciz1 b-variant capture agent;and b) detecting fibrinogen using a fibrinogen detection agent.

As used herein, a ‘Ciz1 b-variant capture agent’ refers to an entitywhich is capable of binding to a Ciz1 b-variant and is used to isolate(i.e. enrich) the fibrinogen/Ciz1 b-variant complex from a sample.Preferably, a ‘Ciz1 b-variant capture agent’ is an entity which iscapable of specifically binding to Ciz1 b-variant. Such specific bindingenables the isolation/enrichment of the fibrinogen/Ciz1 b-variantcomplex from a sample.

The Ciz1 b-variant capture agent may be an antibody or a fragmentthereof, non-protein scaffold, aptamer or Ciz1 b-variant peptide whichspecifically binds fibrinogen.

In particular embodiments, a Ciz1 b-variant/fibrinogen complex may becaptured using an antibody or fragment thereof that binds to the Ciz1b-variant peptide. An antibody which specifically binds a Ciz1 b-variantpeptide may be generated as previously described for antibody 2B(Higgins et al; 2012; PNAS; Nov 6; 109(45):E3128-35).

A ‘Ciz1 b-variant detection agent’ refers to an entity which is capableof binding to a Ciz1 b-variant and is used to bind the fibrinogen/Ciz1b-variant complex after it has been isolated (i.e. enriched) from asample using a capture agent. Preferably, a ‘Ciz1 b-variant detectionagent’ is an entity which is capable of specifically binding to Ciz1b-variant. Such specific binding enables the detection of Ciz1 b-variantfollowing the capture of the fibrinogen/Ciz1 b-variant complex.

The Ciz1 b-variant detection agent may be an antibody or a fragmentthereof, non-protein scaffold, or aptamer which specifically binds Ciz1b-variant.

The Ciz1 b-variant detection agent may be labelled with an entity whichenables it to be detected in an assay as described herein. For example,the Ciz1 b-variant detection agent may be labelled with a fluorescententity or an enzyme-substrate label as described herein.

For example, methods for detecting Ciz1 b-variant may employantibody-based arrays, enzyme linked immunosorbent assays (ELISA),non-antibody protein scaffolds, radioimmuno-assay (RIA), westernblotting, aptamers or mass spectrometry.

An ELISA may be performed according to general methods which are knownin the art. For example, the ELISA may be a sandwich or competitiveELISA.

A sandwich ELISA may comprise the following steps:

-   -   a surface (Le. a microtitre plate well) is prepared to which a        known quantity of capture agent is bound (e.g. a Ciz1 b-variant        capture agent or a fibrinogen capture agent);    -   any nonspecific binding sites on the surface are blocked;    -   the sample comprising the Ciz1 b-variant is applied to the        plate;    -   the plate is washed to remove unbound antigen;    -   a primary antibody which is capable of specifically binding the        fibrinogen or Ciz1 b-variant is added, and binds to antigen;    -   an enzyme-linked secondary antibody is applied as a detection        antibody that bind specifically to the antibody Fc region;    -   the plate is washed to remove the unbound antibody-enzyme        conjugates, before a chemical is added to be converted by the        enzyme into a colour or fluorescent or electrochemical signal;    -   the absorbency or fluorescence or electrochemical signal of the        plate wells is measured to determine the presence and quantity        of antigen.

In particular embodiments, the present methods comprise the step ofcapturing a fibrinogen/Ciz1 b-variant complex using a fibrinogen captureagent or a Ciz1 b-variant capture agent as described herein.

In one embodiment the present methods comprise a sandwich ELISA whichcomprises the steps of capturing a fibrinogen/Ciz1 b-variant complexusing a Ciz1 b-variant capture agent as described herein and detectingthe fibrinogen/Ciz1 b-variant complex using a fibrinogen detection agentas described herein.

A competitive ELISA may comprise the following steps:

-   -   a labelled antibody which specifically binds the Ciz1 b-variant        is incubated in the presence of a sample comprising the Ciz1        b-variant;    -   the bound antibody/antigen complexes are then added to an        antigen-coated well;    -   the plate is washed, so unbound antibody is removed;    -   a secondary antibody, specific to the primary antibody, and        coupled to an enzyme is added;    -   a substrate for the enzyme is added, and the reaction of the        enzyme with its substrate elicits a chromogenic or fluorescent        signal;    -   the reaction is stopped to prevent eventual saturation of the        signal.

Various enzyme-substrate labels are available, e.g. as disclosed in U.S.Pat. No. 4,275,149. The enzyme generally catalyses a chemical alterationof the chromogenic substrate that can be detected. For example, theenzyme may catalyse a colour change in a substrate, or may alter thefluorescence or chemiluminescence of the substrate. Examples ofenzymatic labels include peroxidase such as horseradish peroxidase(HRPO), alkaline phosphatase, beta-galactosidase, glucoamylase,lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase,and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such asuricase and xanthine oxidase), lactoperoxidase, microperoxidase, and thelike. Techniques for conjugating enzymes to antibodies are well known.

Detecting a Ciz1 b-variant peptide may comprise contacting said Ciz1b-variant peptide with an antibody or fragment thereof that binds to theCiz1 b-variant peptide. An antibody which specifically binds a Ciz1b-variant peptide may be generated as previously described for antibody2B (Higgins et al; 2012; PNAS; Nov 6; 109(45):E3128-35).

In some embodiments the b-variant specific antibody may bind to anepitope comprising the EVR sequence shown in positions 36 to 38 of SEQID NO: 11.

In some embodiments, the antibody specifically binds to an amino acidsequence comprising DEEEIEVRSRDIS (SEQ ID NO: 2) but does notspecifically bind to an amino acid sequence comprisingDEEEIEVEEELCKQVRSRDIS (SEQ ID NO: 9).

However, in some embodiments, the sequence bound by a b-variant specificantibody may be present in both Ciz1 exon 14a (SEQ ID NO: 6) and Ciz1exon 14b (SEQ ID NO: 5). In such embodiments, the b-variant specificantibody may be unable to bind to Ciz1 a-variant because the presence ofthe VEEELCKQ sequence (SEQ ID NO: 10) prevents the antibody binding. Inother embodiments, the antibodies may bind to sequences present in bothCiz1 exon 14a and Ciz1 exon 14b, because the sequences are presented ina b-variant specific conformation or structure.

In some embodiments, the b-variant specific antibody may bind to anepitope formed by a Ciz1 b-variant peptide/fibrinogen complex.

Without being bound by theory, the binding of Ciz1 b-variant tofibrinogen may expose epitopes which comprise amino acid sequences whichare common between Ciz1 exon 14a (SEQ ID NO: 6) and Ciz1 exon 14b (SEQID NO: 5) in a conformation which is unique to the fibrinogen/Ciz1b-variant. Such conformational epitopes will therefore not be present inCiz1 a-variant.

Sample

In certain embodiments, the present invention utilises a sample isolatedfrom a subject. This sample may be referred to as a ‘test sample’. Thusthe present methods are typically practiced outside of the human oranimal body, e.g. on a sample that was previously obtained from thesubject to be tested.

The sample may be a blood, urine, saliva or bronchoalveolar lavagesample.

In a preferred embodiment the sample is a blood sample. Suitably, theblood sample may be a whole blood sample. Suitably, the blood sample maybe a blood fraction, for example a plasma sample.

Techniques for collecting blood samples and separating blood fractionsare well known in the art. For instance, vena blood samples can becollected from patients using a needle and deposited into plastic tubes.The collection tubes may, for example, contain anticoagulants,spray-coated silica, or a polymer gel for separation. Plasma can beseparated by centrifugation at 1300 RCF for 10 min at room temperatureand stored in small plastic tubes at −80° C.

In a preferred embodiment the sample is a blood sample, in particular aplasma sample. In a preferred embodiment the blood sample is treatedwith anticoagulants (e.g. heparin) following collection.

Detergent

In one embodiment, the present methods may comprise treating the samplewith a detergent in order to release the Ciz1 b-variant and/or afibrinogen/Ciz1 b-variant complex.

Examples of detergent include, but are not limited to, sodium dodecylsulfate (SDS) and polysorbate 20. For example, the sample may be treatedwith about 1 to 5%, preferably 1 to 2% SDS.

Suitably, the present methods may further comprise treating the samplewith a reducing agent in order to further purify the Ciz1 b-variantand/or a fibrinogen/Ciz1 b-variant complex. Examples of suitablereducing agents include, but are not limited to, Dithiothreitol(DTT)—which may be used at a concentration of about 10 mM DTT, forexample.

Exosomes

The present methods may comprise the step of releasing Ciz1 b-variantand/or a fibrinogen/Ciz1 b-variant complex from the exosomal compartmentof a sample. Suitably, the present methods may comprise the step ofreleasing Ciz1 b-variant peptides from the exosomal compartment of asample.

Exosomes are nanometer-sized (30-100 nm) vesicles found in manybiological fluids, such as urine, blood (in both plasma and serum),ascites, and cerebrospinal fluid. They originate as internal vesicles ofmulti-vesicular bodies (MVBs) in cells and were first described asproducts of circulating blood cells, such as erythrocytes andlymphocytes.

Exosomes are surrounded by a phospholipid membrane containing relativelyhigh levels of cholesterol, sphingomyelin, and ceramide and containingdetergent-resistant membrane domains (lipid rafts) (Mathivanan et al.,2010; J Proteomics 73:1907-1920).

Exosomes are characterized by the presence of proteins involved inmembrane transport and fusion, such as Rab, GTPases, annexins, andflotillin, components of the endosomal sorting complex required fortransport (ESCRT) complex such as Alix, tumor susceptibility gene 101(TSG101), heat shock proteins (HSPs), integrins, and tetraspanins,including CD63, CD81, and CD82 (van der Pol et al.; 2012; 64(3);676-705).

Releasing Ciz1 b-variant and/or a fibrinogen/Ciz1 b-variant complex froman exosomal compartment refers to disrupting the structural integrity ofthe exosome such that Ciz1 b-variant epitopes which were previouslyconcealed within the internal structure of the exosomes are exposed forbinding.

Suitably, releasing Ciz1 b-variant and/or a fibrinogen/Ciz1 b-variantcomplex from an exosomal compartment may refer to treating a sample witha detergent. Examples of detergent include, but are not limited to,sodium dodecyl sulfate (SDS) and polysorbate 20.

In one embodiment, the sample may be treated with a detergent at aconcentration which is suitable to release Ciz1 b-variant and/or afibrinogen/Ciz1 b-variant complex from an exosomal compartment. Forexample, the sample may be treated with about 1 to 5%, preferably 1 to2% SDS.

Exosome Enrichment

The present methods may involve enriching an exosomal fraction from asample.

As used herein, enriching an exosomal fraction is synonymous topurifying or isolating an exosomal fraction.

Exosomes may be purified by methods known in the art such asultracentrifugation (Pisitkun et al (2004) PNAS 101:13368-13373) orultrafiltration (Cheruvanky et al (2007) Am. J. Physiol. Renal Physiol.292:F1657-F1661). Methods are also known involving continuous flowelectrophoresis and chromatography procedures which may precedecentrifugation (Taylor and Gercel-Taylor (2005) Br J Cancer 92:305-311).Cross-flow ultrafiltration may also be used as part of the exosomepurification method (Lamparski et al (2002) J Immunol. Methods270:211-226).

Commercial kits for the isolation of exosomes are also available, forexample the Total Exosome Isolation Kit provided by Invitrogen.

Exosomes can be isolated from a multitude of cell line and body fluidsby combining differential centrifugation, membrane filtration,concentration, rate zonal centrifugation and immunocapture, exosomes(Simpson et al.; Proteomics, 8 (2008), pp. 4083-4099).

Exosomes can be detected and/or purified on the basis of theirexpression of exosomal markers such as tumour susceptibility gene(TSG101), aqua-porin˜2 (AQP2), neuron-specific enolase (NES), annexin V,podocalyxin (PODXL) and CD9. For example, exosomes may be captured on toa microplate, for example by immunoaffinity capture.

Characterization of isolated exosomes is typically performed usingelectron microscopy, FACS, LC-MS/MS, Western blotting or ELISA (Simpsonet al.; as above) and (van Niel et al; J Biochem (Tokyo), 140 (2006),pp. 13-21).

Cancer

In one aspect, the present invention relates to a method of diagnosingcancer in a subject.

As used herein, the term “cancer” or “cancerous” refers to cells havingthe capacity for autonomous growth, i.e., an abnormal state or conditioncharacterized by rapidly proliferating cell growth. The term is meant toinclude all types of cancerous growths or oncogenic processes,metastatic tissues or malignantly transformed cells, tissues, or organs,irrespective of histopathologic type or stage of invasiveness. The term“cancer” includes malignancies of the various organ systems, such asthose affecting, for example, lung, breast, thyroid, lymphoid,gastrointestinal, reproductive and genito-urinary tract, as well asadenocarcinomas which include malignancies such as most colon cancers,renal-cell carcinoma, prostate cancer and/or testicular tumours,non-small cell carcinoma of the lung, cancer of the small intestine andcancer of the esophagus.

Suitably, the cancer may be lung cancer, lymphoma, kidney cancer, breastcancer, liver cancer, bladder cancer, ovarian or thyroid cancer.

In one embodiment, the cancer is lung cancer. The lung cancer may besmall cell lung cancer. The lung cancer may be non-small cell lungcancer.

Subject

The subject may be a human or animal subject. Preferably, the subject isa human.

The subject may have or be at risk of cancer, as described herein. ‘Atrisk of cancer’ refers to a subject who is not showing any symptoms ofthe disease. The subject may have a predisposition for, or be thought tobe at risk of developing, cancer.

Treating

In one aspect the present invention relates to a method for treating asubject with cancer comprising administering to the subject a cancertherapeutic; wherein the subject has been identified as having cancer bya method of the present invention.

In another aspect the present invention provides an anticancer drug foruse in treating cancer wherein the subject has been identified as havingcancer by a method of the present invention. In a further aspect, thepresent invention relates to an anticancer lung cancer drug for use intreating lung cancer wherein the subject has been identified as havinglung cancer by a method of the present invention.

‘Treating’ or ‘to treat’ refers to the therapeutic use of a cancertherapeutic or anticancer agent. Herein the cancer therapeutic oranticancer agent may be administered to a subject having an existingdisease or condition in order to lessen, reduce or improve at least onesymptom associated with the disease and/or to slow down, reduce or blockthe progression of the disease.

Anticancer Drug

An ‘anticancer drug’ is synonymous with a cancer therapeutic and refersto an entity which may be used to treat cancer.

The anti-cancer drug may be a cytokine or haematopoietic factorincluding, but not limited to, IL-1, IL-2, IL-4, IL-5, IL-13, IL-6,CSF-1, M-CSF, GM-CSF, IFNα, IFNβ, IFNγ, IL-10, IL-12, VEGF, bonemorphogenic proteins, FGFs, TNF and TGFβ.

The anticancer drug may be a chemotherapeutic agent, which may be acytotoxic drug. A chemotherapeutic agent contemplated includes, withoutlimitation, alkylating agents, nitrosoureas,ethylenimines/methylmelamine, alkyl sulfonates, antimetabolites,pyrimidine analogs, epipodophylotoxins, enzymes such as L-asparaginase;biological response modifiers such as IFNα, IL-2, G-CSF and GM-CSF;platinium coordination complexes such as cisplatin and carboplatin,anthracenediones, substituted urea such as hydroxyurea, methylhydrazinederivatives including N-methylhydrazine (MIH) and procarbazine,adrenocortical suppressants such as mitotane (o,p′-DDD) andaminoglutethimide; hormones and antagonists includingadrenocorticosteroid antagonists such as prednisone and equivalents,dexamethasone and aminoglutethimide; progestin such ashydroxyprogesterone caproate, medroxyprogesterone acetate and megestrolacetate; estrogen such as diethylstilbestrol and ethinyl estradiolequivalents; antiestrogen such as tamoxifen; androgens includingtestosterone propionate and fluoxymesterone/equivalents; antiandrogenssuch as flutamide, gonadotropin-releasing hormone analogs andleuprolide; and non-steroidal antiandrogens such as flutamide.

The anticancer drug may be a siRNA or shRNA.

Suitably, the anticancer drug may be an anticancer lung cancer drug. Ananticancer lung cancer drug refers to an entity which may be used totreat lung cancer.

Kit

In one aspect, the present invention provides a kit comprising an agentwhich specifically binds fibrinogen and an agent which specificallybinds Ciz1 b-variant.

The agent which specifically binds fibrinogen may be a fibrinogencapture agent as described herein.

The agent which specifically binds Ciz1 b-variant may be a Ciz1b-variant detection agent as described herein.

The terms “comprising”, “comprises” and “comprised of” as used hereinare synonymous with “including”, “includes” or “containing”, “contains”,and are inclusive or open-ended and do not exclude additional,non-recited members, elements or method steps. The terms “comprising”,“comprises” and “comprised of” also include the term “consisting of”.

The invention will now be further described by way of Examples, whichare meant to serve to assist one of ordinary skill in the art incarrying out the invention and are not intended in any way to limit thescope of the invention.

EXAMPLES Example 1 Enrichment of Ciz1 b-Variant in an Exosomal FractionFrom a Blood Sample

The antibody 043 was generated and validated as described previously forantibody 2B (Higgins et al; PNAS 2012; Nov 6; 109(45):E3128-35)). Theantibody was capable of detecting of Ciz-1 b-variant polypeptide bywestern blot in plasma from lung cancer patients (see FIG. 1a ).

Plasma from lung cancer patients was separated into the exosomalcompartment and soluble compartment using Invitrogen Total ExosomeIsolation Kit (Catalog Number 4484450) used as recommended by themanufacturer. Fractions were separated by SDS-PAGE and probed with 043antibody or with ‘generic’ CIZ1 antibodies that recognize an epitopeelsewhere in the protein (data shown in FIG. 1b is generated withNB100-74624 (Nov4), Novus Biologicals).

The Ciz1 b-variant epitope containing species migrated with relativemobility of ˜70 kDa. The Ciz1 b-variant epitope containing speciesco-fractionated with the exosomal fraction in lung cancer patients (seeFIG. 1b , red and blue arrows indicate lanes showing differentpartitioning of Ciz1 isoforms). In rare patients, the Ciz1 b-variantepitope containing species partly partitions with the non-exosomalfraction (type 2, FIG. 1c ). Plasma from these patients gave a positivesignal in Ciz1 b-variant sandwich ELISA without any pre-treatment todissolve exosomes. For patients whose Ciz1 b-variant epitope containingspecies is wholly in the exosome fraction, the Ciz1 b-variant epitopemay be accessed by treating plasma (type 1) with detergent prior toassay (FIG. 1e,f ).

Example 2 Reconstitution of Epitope From Synthetic b-Variant Peptidesand Fibrinogen

The antibody 2B (FIGS. 3a ) and 043 (FIG. 1a ) recognise Ciz1 b-variantin the region of the unique junction between exon 14b and 15. They donot react with a species with relative mobility of ˜70 kDa in normalplasma (FIG. 3B, C), but do weakly react with synthetic b-variantpeptide (FIG. 3B, C). However when combined, synthetic b-variantpeptides and normal plasma generate a new, highly reactive species of˜80 kDa. Similar results are obtained with a synthetic b-variant peptideof a different length (FIG. 3e ), quantitatively generating epitope(FIG. 3f ).

Mobility of endogenous b-variant epitope in plasma from cancer patientsis sensitive to reducing agent, migrating at ˜340 kDa in the absence ofDTT and at ˜70 kDa in the presence of DTT (FIG. 4a ). Excision of the340 kDa complex (FIG. 4b ), followed by further separation intoconstituent polypeptides upon reduction shifts the epitope to ˜70 kDa(FIG. 4c ). Excision and elution of this band identified fibrinogenalpha chain by mass spectrometry (FIG. 4d ).

Incubation of purified fibrinogen complex (FIG. 5a ) with syntheticb-variant peptides generates a b-variant epitope with mobility similarto the fibrinogen complex (FIG. 5a ). Comparison with a-variantpeptides, or carboxylated derivatives of b-variant peptides, or withdifferent lengths of b-variant peptides showed that i) reconstitution ofepitope is specific to b-variant containing peptides, ii) carboxylationof certain E residues near the splice junction affects reconstitution ofepitope, iii) b-variant junction containing peptides of differentlengths may constitute the epitope that naturally exists in humanplasma.

Example 3 Characterisation of Ciz-1 b-Variant as a Biomarker

Analysis of CIZ1b was carried out with anti-peptide rabbit polyclonalantibody 2B, which was raised against a short unique peptide spanningthe alternatively spliced 14b-15 junction (FIG. 7A), with sequenceDEEEIEVRSRDIS (SEQ ID NO: 2—junction indicated by underlined valine).The strategy for generation, validation and purification was describedpreviously (Higgins et al., as above). The western blot reactivityprofile for 2B includes a 65-70 kDa entity in SDS-PAGE of plasma fromlung cancer patients, plus a 55 kDa entity in plasma from all people(FIG. 7B). The cancer-specific 65-70 kDa species is the CIZ1b biomarkerthat is referred to here and by Higgins et al. In contrast to plasma,this protein could not be detected in serum samples from the same lungcancer patients (FIG. 7C), suggesting that CIZ1b biomarker issequestered in coagulated blood. Notably the 55 kDa generic bandco-migrates with CIZ1 spec7), identifying the 55 kDa species as avariant or proteolytic fragment of full-length hCIZ1 (which has apredicted full length MW of 99 kDa, NCBI Reference Sequence:NM_012127.2). The width of the bands detected via exon 8 and exon 17 isdifferent, possibly because the exon 8 epitope lies within analternatively spiced region, while detection via exon 17 would revealspecies with or without alternative splicing at exon 8. Thus at leastone form of the CIZ1 protein is present in the circulatory system incancer and non-cancer plasma samples alike, suggesting it to be part ofnormal physiology.

Exon 17 and exon 8 epitopes are not present in the 65-70 kDa speciesrecognised by CIZ1b antibody 2B in cancer patients, which means thatsequences both upstream and downstream of the CIZ1b epitope are missingfrom this protein. The data suggest that the 65-70 kDa species is eithera complicated CIZ1 splice variant, or an SDS-stable complex between aCIZ1b fragment of CIZ1 and a carrier protein (FIG. 7D).

A further set of rabbit antibodies was generated using the sameimmunisation protocol as for 2B (see Higgins et al., as above). Thisyielded antibody 043, which cleanly and uniquely reacts with the 65-70kDa band in cancer patients (FIG. 7B). Thus 043 provides a detectiontool with applications in other assay formats, including ELISA. The lackof recognition of the 55 kDa species suggests less contribution ofjunction flanking sequences to its epitope compared to 2B, and suggeststhat the 55 kDa species does not contain the CIZ1b junction.

Direct comparison of the ability of these two antibodies to discriminatelung cancer patients by western blot shows them both to bediscriminatory, with a high degree of correlation (FIG. 7E), thoughnotably they do not share exactly the same profile across plasma testset. Thus, there is some variation in their epitopes but a commonability to discriminate patients with lung cancer from those without.There is also a small advantage in integrating their output to maximiseselectivity.

Example 4 Stability of the Ciz-1 b-Variant Biomarker

The data support the assertion that CIZ1b is a potent circulatingbiomarker for lung cancer with significant potential for application inclinical practise. To realise this potential it must be robust enough towithstand variations in sample processing and collection regimes thatoccur in busy hospitals. Therefore, the stability of the 65-70 kDacancer-selective CIZ1b species in plasma and whole blood was tested, andcompared with the 55 kDa species, using antibody 2B. The data showstability in isolated plasma over at least 6 hours when incubated at 37°C., with gradual decay in both species thereafter. Similarly there wasrelatively little change during an hour at temperatures up to 50° C.,and stability over at least 3 freeze-thaw cycles. Furthermore, whenwhole blood was left on the bench for up to 24 hours prior tofractionation there was no loss of signal, all of which indicate it tobe a robust biomarker suitable for application in a range of clinicalsettings.

Example 5 Dissociation of the Ciz-1 b-Variant Biomarker Complex

Under native conditions both 2B and 043 detect a complex in excess of720 kDa that co-migrates with an abundant protein in plasma (FIG. 8A),but there is very little discrimination between cancer and non-cancerplasmas. Upon further separation through a second dimension underdenaturing conditions the cancer-specific 65-70 kDa species is revealedand (for 2B) separated from the 55 kDa generic band (FIG. 8B). Thuscancer-specific detection of CIZ1b in a completely native immunoassayformat is unlikely even with 043. The three major denaturing influences(heat, SDS, reducing agent) that reveal cancer-specific signal and wereevaluated for their effect on the mobility and discrimination of CIZ1bby 043. This showed that 1% SDS shifts the major reactive speciesfrom >720 kDa in native gel, to approximately 340 kDa (FIG. 8C, right),revealing cancer-specific signal, as well as an unspecific band at ˜200kDa in both cancer and non-cancer samples. Increasing the incubationtemperature has a detrimental effect on the cancer-specific species,with maximum differential achieved at 37° C. Inclusion of reducing agentfurther shifted the mobility of the cancer specific signal to theexpected position of 65-70 kDa (FIG. 8C, left).

Notably, higher concentrations of reducing agent resulted in loss ofCIZ1b signal (FIG. 8D,E). Taken together these data suggest that the65-70 kDa entity is itself a complex that is resistant to standardSDSPAGE denaturing conditions (10 mM DTT) but not more aggressivetreatments (500 mM DTT), and that it is normally present within a higherorder complex of 340 kDa, which is itself released by SDS from a stillbigger complex in excess of 720 kDa (illustrated in FIG. 9A). In fact,fractionation of plasma to enrich for the exosomal compartmentdifferentially partitioned the 55 kDa species (detected via exon 17) and65-70 kDa species (detected via 043), with the 65-70 kDa speciesdetected exclusively in the pellet, which is a complex fraction thatincludes exosomes.

Example 6 Identification of the Ciz-1 b-Variant Biomarker Complex

These observations enable a two-step gel-purification strategy for the65-70 kDa CIZ1b band. First, the 340 kDa species was isolated from twoindependent lung cancer patient plasmas (FIG. 10A), followed by furtherseparation in the presence of reducing agent (FIG. 10B). The 65-70 kDaband corresponding to the cancer-specific western blot signal wasexcised and digested with either trypsin or Asp-N endoprotease, foridentification by mass spectrometry (MS). For both enzymatic digestionsand two patients, Mascot database searching returned a singlesignificant identification (P<0.05), corresponding to Uniprot P02671;human fibrinogen alpha chain. The endogenous fibrinogen molecule is ahexameric glycoprotein with a pre-cleavage formula mass of 326 kDa andrelative mobility of ˜340 kDa, comprised of two sets of three differentchains (α, β, and γ) of 67, 51 and 45 kDa respectively.

Notably, CIZ1b was not identified in the 65-70 kDa band after digestionwith either enzyme. To determine whether a diagnostic CIZ1b fragmentcould be observed by MS if present, a short synthetic CIZ1b peptide(b13) and long CIZ1b peptide (b66, Table 2) were digested with Asp-N,which generates the diagnostic fragment DEEEIEVRSR (SEQ ID NO: 16) bycutting either side of the CIZ1b exon junction. The peptide waspositively identified post digestion of b13 and b66 by MALDI-MS/MS,though at low intensity relative to other peptides in the digest.Further analysis was performed by LC-MS/MS using both data dependentacquisition (DDA) and targeted selection reaction monitoring (SRM),which focused the analysis to cycle through fragmentation of theexpected 2+ and 3+ ions of DEEEIEVRSR (SEQ ID NO: 16), excluding allother precursors, to maximize specificity and sensitivity for thisanalyte. DEEEIEVRSR (SEQ ID NO: 16) was identified in both the DDA andSRM analyses, indicating that the diagnostic peptide is observable by MSwhen excised from chemically synthesized peptide (SRM chromatogram).

However, when the same peptide was spiked into normal human plasma toreconstitute CIZ1b, the diagnostic DEEEIEVRSR (SEQ ID NO: 16) peptidewas not detected in the excised 65-70 kDa band. Lack of identificationcould reflect: i) secondary modifications that alter the expected massof the CIZ1b diagnostic peptide or impair enzymatic digestion, ii) themore abundant proteins in the sample, primarily fibrinogen alpha chain,causing ion suppression or iii) absolute abundance falling below thelimit of detection for the instrumentation after the losses incurredfrom the PAGE and digestion steps. It should be noted that the peptideis highly acidic, which is likely to reduce ionization efficiency inpositive-mode MS, so that even with SRM a small drop in abundance couldfall below the limit of detection. It was concluded if the spikedpeptide could not be reliably detected at physiologically relevantlevels it was unlikely to be possible to detect endogenous CIZ1b, and sofollowed alternative approaches were followed.

TABLE 2 Designation Sequence Short CDEEEIEVRSRDIS-NH2 CIZ1b(SEQ ID NO: 22) (b13) Super  CEIEVRSR-NH2 short (SEQ ID NO: 17) CIZ1b(b7) Short CDEEEIEVEEELCKQVRSRDISR-NH2 CIZ1a (SEQ ID NO: 18) (a22) LongEIAGQDEDHFITVDAVGCFEGDEEEEE CIZ1b DDEDEEEIEVRSRDISREEWKGSETYS peptidePNTAYGVDFLVP (b66) (SEQ ID NO: 11) Long EIAGQDEDHFITVDAVGCFEGDEEEEECIZ1a DDEDEEEIEVEEELCKQVRSRDISREE peptide WKGSETYSPNTAYGVDFLVP (a74)(SEQ ID NO: 19) GLA3 CDEEγIEVRSRDIS-NH2 (SEQ ID NO: 14) GLA4CDEγEIEVRSRDIS-NH2 (SEQ ID NO: 23) GLA6 CDγγγIγVRSRDIS-NH2(SEQ ID NO: 15) Exon 17 C-TSSGRPPSQPNTQDKTPSK (SEQ ID NO: 20)C-TARPSQPPLPRRSTRLKT (SEQ ID NO: 21) The 8 amino-acids spliced out ofCIZ1b are underlined in non-alternatively spliced sequences, and thejunction created by their absence is indicated in CIZ1b sequences by aV. Where indicated peptides contain an additional N-terminal cysteineresidue, and amidation of the C-terminus. Carboxylated glutamic acidresidues are indicated by γ.

Example 7 Epitope Reconstitution of the Ciz-1 b-Variant BiomarkerComplex

In order to confirm that the 65-70 kDa cancer-specific epitope doesindeed contain CIZ1b sequences, an epitope reconstitution strategy wasadopted. To achieve this, a pair of synthetic peptides spanning theexon14/15 junction (CIZ1b13, and CIZ1a22 which includes thealternatively spliced intervening sequence, Table 2) were incubated withnormal human plasma (FIG. 10C), and the output compared to lung cancerpatient plasma. The CIZ1b peptide, but not CIZ1a, generated 043 and2B-reactive epitope of comparable mobility to the endogenous epitope (6570 kDa), which is far in excess of the relative molecular mass of freepeptide and not present in either of the individual components of thereaction (peptide lanes 6, 7, or plasma lane 3). This suggests thatCIZ1b peptide forms an SDS/DTT-resistant complex with a carrier proteinin human plasma, recreating the CIZ1b biomarker recognised by 2B and043. Since no mobility difference between endogenous (cancer plasmalane 1) and reconstituted epitope was detected, the data also imply thatthe CIZ1b fragment contributing to the 65-70 kDa cancer-specific speciesis very short, in the order of 10-20 amino acids in length. Furthermore,individual molecules may not necessarily have uniform molecularboundaries, possibly accounting for the often-diffuse nature of theband.

A molar equivalent of the much longer CIZ1b peptide (b66, Table 2)reconstituted reactive epitope less efficiently, and was detectable onlyby 2B (FIG. 10C lane 2, and at higher concentration). With this peptidethe reconstituted signal migrated with greater relative molecular mass,at ˜80 kDa, consistent with the increased size of the contributingpeptide. Importantly reconstituted CIZ1b epitope, like endogenousepitope, resists standard denaturing SDS-PAGE conditions (10 mM DTT, 2%SDS, 90° C.), but is dissociated by a more aggressive reducingenvironment. Taken together the data suggest that, in vivo, the cancerspecific epitope is composed of a relatively short fragment of CIZ1encompassing the exon14b/15 junction, mounted on fibrinogen alpha chain,which itself makes up most of the mass of the 65-70 kDa species. Tofurther confirm this, epitope reconstitution experiments were performedwith purified human fibrinogen (FIG. 10D). For both antibodies, reactiveepitope was generated at the mobility of fibrinogen complex (340 kDa innon-reducing gels) and the specificity of the signal was confirmed bylack of reactivity with equivalent CIZ1a peptide. Direct ELISA usinghuman fibrinogen complexed with molar equivalents of CIZ1a and CIZ1bpeptides (FIG. 10E), confirmed i) the specificity of epitopereconstitution with peptide b13 over a22, ii) negligible signal withfibrinogen alone, and iii) greater reactivity when peptide is mounted onfibrinogen. Moreover, inclusion of the very short CIZ1b peptide b7(Table 2) revealed differences between 2B and 043; 2B recognises b7while 043 does not in a manner that is unrelated to the presence offibrinogen. The existence of CIZ1b epitope in a complex with fibrinogenis consistent with the earlier observations that the biomarker isdepleted from serum samples isolated from clotted blood (FIG. 7C).

Example 8 CIZ1b Immunoassay

Based on knowledge of the composition of CIZ1b biomarker, a sandwichimmunoassay format capable of quantitative detection in partiallydenatured plasma was designed. Antigen capture with CIZ1b antibody 043from 5 ul of plasma and detection via fibrinogen alpha chain, generateda cancer-specific signal. Specifically, results by western blot with 043generated ROC AUC 0.823 (P=0.0002) and by ELISA with 043/anti-fibrinogenof 0.830 (P=0.0007, FIG. 11A,B). Thus, the two methods generate verysimilar outcomes, and the data is significantly correlated.

This data set, which represents patients with a wide range of conditionspresenting at York Hospital respiratory medicine clinics, was also usedto evaluate the contribution of the CIZ1b component of the assay, sincefibrinogen alone has been reported to have some potential as a biomarkerfor lung cancer (AIlin et al.; 2016; Int J Cancer 139(7):1493-1500).Quantitative detection of fibrinogen in the linear range returned somediscriminatory capability across this set, though relatively poor ROCAUC of 0.628 (p=0.225). This is consistent with western blot evaluationof fibrinogen, which indicated little difference between lung cancerpatients and those without malignant disease. Thus capture of CIZ1b isthe element that contributes the majority of the cancer selectivity tothis assay. The specificity of the configuration was further tested byanalysing the sample set after capture with 043, but substitutinganti-fibrinogen detector antibody with anti-human IgG (FIG. 11D).Results indicate no discrimination between patients and control plasmas,confirming that anti-fibrinogen is an informative detector reagent thatspecifically detects a cancer-selective complex retrieved via CIZ1b.Finally, measurement of total IgG levels across set B by direct ELISA(FIG. 11E) was used to normalize the output generated by 043/fibrinogen,leading to a marginal improvement in discrimination and ROC AUC 0.845(p=0.0002, FIG. 11F).

The CIZ1b-fibrinogen alpha chain complex ELISA was performed as asandwich ELISA after partial denaturation of 5 ul of plasma in 100 ul of0.5% tween20, 1% SDS in PBS, supplemented with 0.5 mM PMSF (proteaseinhibitors) and incubation at 20° C. for 30 mins with repeatedvortexing. Plates (Nunc Maxisorb 442404) were coated with 1 ug of 043CIZ1b pAb protein A fraction, overnight at 4° C. in 100 ul of 50 mMcarbonate pH 9.6, then blocked for two hours at room temperature with300 ul of filtered 1% BSA in PBS. After three washes in 300 ul of 0.05%tween20 in PBS, analyte was added for 2 hours at room temperature withgentle shaking. After fives washes, captured complex was detected withsheep anti-fibrinogen pAb (AF4786 R&D systems) followed by anti-sheepHRP (Jackson ImmunoResearch 213-032-177), and results developed using100 ul Sure Blue (KPL 520001) with detection at A450 nm using a FLUOstarOPTIMA plate reader (BMG Labtech).

For measurement of fibrinogen capture antibody was chicken IgY (1ug/well, Genway 15-288-22856), and analyte concentration was reducedfive orders of magnitude by serial dilution. For measurement of humanIgG in direct ELISA, 0.5 ul of plasma was coated directly onto plates in100 ul of 50 mM carbonate buffer pH 9.6, blocked, washed and detectedwith anti-IgG (Jackson Immuno Research 709-035-149). For direct ELISA ofsynthetic analyte, 0.5 ug purified fibrinogen (Sigma F3879) complexedwith 100 pmol peptide was coated onto plates in 100 ul of 50 mMcarbonate pH 9.6 as above, probe with 043 or 2B and detected withanti-rabbit HRP (Jackson ImmunoResearch).

Thus, the present sandwich ELISA format is suitable for high-throughputapplication on hospital platforms, and can reliably quantify circulatingCIZ1b biomarker, and can be used to identify patients with early stagelung cancer.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

1-7. (canceled)
 8. A method of detecting cancer in a subject whichcomprises detecting a Ciz1 b-variant peptide in a sample from thesubject, wherein the presence of Ciz1 b-variant peptide in the sampleindicates that the subject has cancer and wherein the method comprisesthe step of capturing a fibrinogen/Ciz1 b-variant peptide complex fromthe sample.
 9. A method of detecting cancer in a subject which comprisesdetecting a Ciz1 b-variant peptide in a sample from the subject, whereinthe presence of Ciz1 b-variant peptide in the sample indicates that thesubject has cancer and wherein the Ciz1 b-variant peptide is thesequence shown as SEQ ID NO: 11 or a fragment thereof which comprisesEVR as shown in positions 36 to 38 of SEQ ID NO:
 11. 10. A methodaccording to claim 8 which comprises the following steps: (a) capturinga fibrinogen/Ciz1 b-variant complex using a fibrinogen capture agent;and (b) detecting Ciz1 b-variant using a Ciz1 b-variant detection agent;or (a) capturing a fibrinogen/Ciz1 b-variant complex using a Ciz1b-variant capture agent; and (b) detecting fibrinogen using a fibrinogendetection agent.
 11. The method according to claim 10 wherein the Ciz1b-variant capture agent is an antibody or a fragment thereof, or anon-protein scaffold which specifically binds Ciz1 b-variant.
 12. Themethod according to claim 10 wherein the Ciz1 b-variant capture agent isan antibody which specifically binds Ciz1 b-variant.
 13. The methodaccording to claim 8 wherein the step of detecting the Ciz1 b-variantpeptide or fibrinogen employs antibody-based arrays, enzyme linkedimmunosorbent assays (ELISA), non-antibody protein scaffolds,radioimmuno-assay (RIA), western blotting, aptamers or massspectrometry.
 14. The method according to claim 8, further comprisingthe step of releasing a Ciz1 b-variant peptide and/or a fibrinogen/Ciz1b-variant complex from an exosomal compartment of the sample.
 15. Themethod according to claim 14 wherein the Ciz1 b-variant peptide and/orfibrinogen/Ciz1 b-variant complex is released from the exosomalcompartment by detergent treatment.
 16. The method according to claim14, further comprising the step of enriching the exosomal fraction fromthe sample.
 17. The method according to claim 16, wherein the step ofenriching the exosomal fraction comprises ultracentrifugation,ultrafiltration, continuous flow electrophoresis, chromatography,precipitation or cross-flow ultrafiltration.
 18. The method according toclaim 10 wherein the cancer is selected from lung, lymphoma, kidney,breast, liver, bladder, ovarian or thyroid cancer.
 19. The methodaccording to claim 18 wherein the cancer is lung cancer.
 20. A method oftreating a subject with cancer comprising administering to the subject acancer therapeutic; wherein the subject has been identified as havingcancer by the method as defined in claim
 10. 21-23. (canceled)
 24. Amethod of detecting a Ciz1 b-variant in a sample which comprises thestep of releasing a Ciz1 b-variant and/or a fibrinogen/Ciz1 b-variantcomplex from an exosomal compartment of the sample by treating thesample with detergent and detecting if the Ciz1 b-variant is present inthe sample.
 25. The method according to claim 24 wherein the Ciz1b-variant is in a complex with fibrinogen.
 26. The method according toclaim 24 which comprises the step of enriching the exosomal fractionfrom the sample prior to releasing the Ciz1 b-variant.
 27. The methodaccording to claim 26, wherein the step of enriching the exosomalfraction comprises ultracentrifugation, ultrafiltration, continuous flowelectrophoresis, chromatography, precipitation or cross-flowultrafiltration.
 28. A kit comprising an agent which specifically bindsfibrinogen and an agent which specifically binds Ciz1 b-variant.
 29. Akit according to claim 28 wherein the fibrinogen is fibrinogen alphachain.
 30. A kit according to claim 28 wherein the agent whichspecifically binds fibrinogen and/or the agent which specifically bindsCiz1 b-variant is an antibody or a fragment thereof, a non-proteinscaffold or an aptamer.